RESEARCH ARTICLE

Balanced Rac1 activity controls formation and maintenance ofneuromuscular acetylcholine receptor clustersYanyang Bai1,*, Daji Guo1, Xiaoyu Sun1, Genyun Tang2, Tailin Liao3, Yinghui Peng1, Junyu Xu3 and Lei Shi1,‡

ABSTRACTRac1, an important Rho GTPase that regulates the actin cytoskeleton,has long been suggested to participate in acetylcholine receptor(AChR) clustering at the postsynaptic neuromuscular junction.However, how Rac1 is regulated and how it influences AChRclusters have remained unexplored. This study shows that breakingthe balance of Rac1 regulation, by either increasing or decreasing itsactivity, led to impaired formation and maintenance of AChR clusters.By manipulating Rac1 activity at different stages of AChR clustering incultured myotubes, we show that Rac1 activation was required for theinitial formation of AChR clusters, but its persistent activation led toAChR destabilization, and uncontrolled hyperactivation of Rac1 evencaused excessive myotube fusion. Both AChR dispersal and myotubefusion induced by Rac1 were dependent on its downstream effectorPak1. Two Rac1 GAPs and six Rac1 GEFs were screened and foundto be important for normal AChR clustering. This study reveals that,although general Rac1 activity remains at low levels during terminaldifferentiation of myotubes and AChR cluster maintenance, tightlyregulated Rac1 activity controls normal AChR clustering.

KEY WORDS: Neuromuscular junction, Acetylcholine receptor,Agrin, Rho GTPase, Rac1, Actin

INTRODUCTIONThe formation, dispersal and maintenance of acetylcholinereceptors (AChRs) at the neuromuscular junction (NMJ) require ahighly dynamic control of local cytoskeleton (Sanes and Lichtman,1999, 2001; Shi et al., 2012; Wu et al., 2010). In general, anchorageof AChR clusters to the cytoskeleton, mediated by a plethora ofscaffold proteins, such as rapsyn and the dystrophin–glycoproteincomplex, determines the stability of the receptor clusters (Gautamet al., 1995; Gawor and Proszynski, 2017; Jacobson et al., 2001).However, the local cytoskeleton environment, such as actinpolymerization/depolymerization, is crucial for the dynamicaddition/removal of AChRs at the synaptic sites (Dai et al., 2000;

Dobbins et al., 2006). For example, addition and loss of AChRs areboth found to occur at the sites of actin polymerization, suggestingthat elevated actin dynamics is associated with poor stability or highturnover of AChRs (Basu et al., 2015; Lee et al., 2009; Proszynskiet al., 2009).

The Rho family of small GTPases, including RhoA, Rac1 andCdc42, is one of the main regulators of actin cytoskeleton and isfound to have important roles in AChR clustering mediated by agrin,a factor secreted by neurons, which aids postsynaptic organization ofneuromuscular junctions and other signals, such as laminin and Wnt(Henriquez et al., 2008; Linnoila et al., 2008; Nizhynska et al., 2007;Weston et al., 2003, 2000). The activity of Rho GTPases arecontrolled by two classes of regulatory protein, guanine nucleotideexchange factors (GEFs) and GTPase-activating proteins (GAPs),which activate and inactivate Rho GTPases, respectively (Bai et al.,2015; Rossman et al., 2005; Tcherkezian and Lamarche-Vane,2007). In vivo activation of RhoA through the RhoA GEF ephexin1(Ngef) selectively removes receptors within the AChR clusters,leading tomorphological maturation of the clusters (Shi et al., 2010a,b). Rac1 activity has long been suggested to mediate the formationof AChR clusters, but may also participate in the endocytosis ofAChRs (Henriquez et al., 2008; Kumari et al., 2008; Weston et al.,2003, 2000). Nonetheless, how Rac1 activity influences AChRclusters in vivo, and which GEFs and GAPs are involved in Rac1regulation during AChR clustering has not been explored.

In our present study, we show that global expression ofconstitutively active Rac1 in cultured myotubes caused excessivemyotube fusion. Interestingly, only transient Rac1 activation wasinduced following addition of agrin, and both prolonged Rac1activation as well as Rac1 inhibition impair formation of AChRclusters. Moreover, AChR clusters are smaller and easier todestabilize after Rac1 activation. We further found that the serine/threonine-protein kinase Pak 1 (Pak1) is a major downstreameffector mediating the effect of Rac1 on myotube fusion and AChRcluster dispersal. Finally, several Rac1 GEFs and GAPs wereidentified to be important for normal AChR cluster formation.

RESULTSConstitutive activation ofRac1 in culturedmyotubes leads toabnormal myotube fusionTo study the function of Rac1 in the regulation of AChR clustering,we transfected the mRNAs of Rac1 wild-type (Rac1-WT),constitutively active Rac1 (Rac1-G12V) or dominant-negativeRac1 (Rac1-T17N) in cultured C2C12 myotubes. By usingmRNAs, we were able to achieve high transfection efficiency(>90% of the myotubes were transfected) and rapid expression (2 hpost transfection; Fig. S1A,B) (Shi et al., 2010a). Surprisingly,myotubes expressing Rac1-G12V fused together extensively,exhibiting more ‘myopatches’ instead of myotubes (Fig. 1A,B).Rac1-Q61L, another constitutively active Rac1 mutant that locksRac1 at the GTP-bound state by disrupting its GTPase activity,Received 17 January 2018; Accepted 2 July 2018

1JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research,College of Pharmacy, Jinan University, Guangzhou 510632, Guangdong, China.2Department of Medical Genetics, Hunan Provincial Key Laboratory of DongMedicine, Hunan University of Medicine, Huaihua 418000, Hunan, China.3Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministryof Health, Zhejiang Province Key Laboratory of Neurobiology, Zhejiang UniversitySchool of Medicine, Hangzhou 310058, Zhejiang, China.*Present address: Centre for Gene Editing Brain Disease Models, The BrainCognition and Brain Disease Institute, Shenzhen Institutes of AdvancedTechnology, Chinese Academy of Sciences, 1068 Xueyuan Boulevard, Xili,Nanshan, Shenzhen 518055, China.

‡Author for correspondence (sophielshi80@gmail.com)

Y.B., 0000-0002-9923-6843; D.G., 0000-0002-6316-3443; X.S., 0000-0001-7076-5648; G.T., 0000-0001-7936-1594; T.L., 0000-0001-8728-1033; Y.P., 0000-0002-6714-5023; L.S., 0000-0001-8695-3432

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© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs215251. doi:10.1242/jcs.215251

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induced myotube fusion similar to that of Rac1-G12V (Fig. 1A,B).Decreasing the amount of Rac1-G12V or Rac1-Q61L mRNA by asmuch as 25 times (0.02 μg) still induced myotube fusion (Fig. 1C).By contrast, increasing the amount of Rac1-WT mRNA 10 times(5 μg) did not affect the myotube width (Fig. S2), suggesting thatthere are highly regulated mechanisms for Rac1 inhibition in maturemyotubes. To study how constitutive activation of Rac1 affectsmyotube morphology, we labeled active Rac1 by transfectingmRNA of the Rac1-binding domain (PBD) of Pak1 tagged to that ofyellow fluorescent protein (YFP-PBD), a probe that specificallybinds to the active GTP-bound form of Rac1 (Hoppe and Swanson,2004). Filamentous actin (F-actin) was stained with Rhodamine-phalloidin. In control myotubes, we hardly observed signals of

active Rac1, and F-actin signals were generally diffuse with higherdistribution at the myotube surface (Fig. 1D, left). In Rac1-G12V-transfected myotubes, however, active Rac1 was strongly labeled atthe junctional surface of fusing myotubes, where it stronglycolocalized with concentrated F-actin (Fig. 1D, right). Whenagrin was added to induce AChR clustering, the number ofclusters found in Rac1-G12V transfected myotubes was increasedby more than double (Fig. 1E,F). However, the average intensityof individual clusters labeled with α-bungarotoxin (α-BTX)conjugated Alexa Fluor555 in Rac1-G12V myotubes was onlyhalf of that observed in control myotubes (Fig. 1G), suggestinga reduced AChR density. The average cluster area was notsignificantly changed, although very large clusters (>300 μm2)

Fig. 1. Hyperactivation of Rac1 incultured myotubes leads toabnormal myotube fusion.(A) Different Rac1 plasmids weretransfected in cultured C2C12myotubes that had differentiated for3.5 days. 24 h later, myotubes werestained with anti-myosin heavy chain(MHC) antibody. Scale bar: 100 μm.(B) The width of myotubes wasquantified as an indication of myotubefusion. Both constitutively active Rac1plasmids Rac1-G12V (G12V) andRac1-Q61L (Q61L) caused severemyotube fusion. ***P<0.001, Rac1-G12V or Rac1-Q61L vs Ctrl, Rac1-WT or Rac1-T17N, one-way ANOVAfollowed by Bonferroni multiplecomparison test. (C) Differentamounts of Rac1-G12V or Rac1-Q61L (0.02–0.5 μg) were tested formyotube fusion. *P<0.05, **P<0.01,***P<0.001, Rac1-G12V or Rac1-Q61L vs Ctrl, one-way ANOVAfollowed by Bonferroni multiplecomparison test. (D) Constitutivelyactive Rac1 colocalized with F-actinand the fusion sites of myotubes.YFP-PBD was transfected togetherwith Rac1-G12V into C2C12myotubes. F-actin was stained byRhodamine-phalloidin. Top panels:YFP-PBD signals; middle panels:F-actin signals; bottom panels:Merged signals of YFP-PBD (green)and F-actin (red). Scale bar: 50 μm.(E) Hyperactivation of Rac1 disruptsAChR clustering. Rac1-G12V wastransfected into C2C12myotubes andagrin was added for 8 h. AChRclusters were stained with AlexaFluor555-conjugated to α-BTX. Scalebar: 50 μm. (F–H) Numbers of AChRclusters (F), fluorescent intensity(G) and size of cluster area (H) werequantified. ***P<0.001, Student’st-test; 10 images were quantified ineach experiment. All data shown inthis figure are presented as mean±s.e.m. from at least threeindependent experiments.

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were occasionally observed in Rac1-G12V myotubes (Fig. 1H).Together, these findings suggest that Rac1 is normally inhibited inmature myotubes, and that constitutive activation of Rac1 leads toelevation of actin polymerization at the myotube surface, as well asto excessive myotube fusion and disruption of AChR clustering.Because the dramatic change of AChR clusters is possibly aconsequence of the myotube fusion instead of a direct regulation byRac1 G12V, we only used Rac1-WT for activation of Rac1 in oursubsequent studies of AChR clustering.

Rac1 is transiently activated at the initial stage of AChRclustering and rarely colocalized with AChR clustersBy adding agrin to C2C12 myotubes for different durations, weobtained a time-course pattern of AChR clustering. The first 2 h ofagrin treatment appeared to be the initial stage of AChR clustering,as there was a high number of AChR microclusters (<5 μm2) and alow number of full-sized clusters (≥5 μm2; Fig. 2A,B). From 2–8 hof agrin treatment, the number of microclusters decreased by >50%,concomitantly with the dramatic 5-fold increase of full-sizedclusters (Fig. 2A,B). The numbers of both full-sized clusters andmicroclusters remained relatively unchanged between 8 and 16 h(Fig. 2A,B), suggesting that agrin-induced AChR clustering reachesa maximum at 8 h, remaining stable afterwards. Interestingly, agrininduced a rapid and transient activation of Rac1, which peaked at15 min and decreased to almost basal level at 60 min (Fig. 2C). Thisis consistent with previous reports (Linnoila et al., 2008; Westonet al., 2003), suggesting that Rac1 is only transiently activated atthe initial stage of AChR clustering. To study the cocalization ofactive Rac1 relative to AChR clusters, we transfected myotubes withmRNAs of Rac1-WT or Rac-T17N, together with mRNA of YFP-PBD, followed by addition of agrin for 1.5 h. Rac1-WT, indeed,increased the number of active Rac1 clusters, whereas Rac1-T17N

diminished active Rac1 clusters (Fig. 2D). Surprisingly, active Rac1clusters were infrequently detected on the myotube surface oradjacent to AChR clusters, but rarely colocalized with AChRclusters (Fig. 2D). These observations suggest that Rac1 activationis associated with the dynamics of AChRs, such as AChR addition,movement or dispersal.

Initial formation of AChR clusters requires Rac1 activationWe first studied whether changes of Rac1 activity affect differentstages of agrin-induced AChR cluster formation. Rac1-WT or T17NmRNA was transfected into myotubes, and agrin was added for1.5 h or 8 h to induce initial and maximal clustering, respectively(Fig. 3A). Rac1-WT significantly increased the initial formation ofmicroclusters (Fig. 3B,C), but Rac1-T17N inhibited the initialformation of both microclusters and full-sized clusters (Fig. 3B–E).When agrin was added for 8 h, Rac1-WT still increasedmicroclusters, but both Rac1-WT and Rac1-T17N reduced thenumber of full-sized AChR clusters (Fig. 3F–I). These resultssuggest that, although Rac1 activation is required for the initialformation of AChR clusters, its prolonged activation preventsnormal formation of full-sized clusters.

Stabilization of AChR clusters depends on Rac1 inhibitionAs Rac1 is only transiently activated upon treatment with agrin, weintroduced Rac1-WT and Rac1-T17N into myotubes after the initialphase of AChR clustering, i.e. transfection was performed after 1 h ofagrin treatment when Rac1 activity had returned to basal levels(Fig. S3A). Interestingly, inhibition of Rac1 had almost no effect onAChR clustering (Fig. S3B–E). Activation of Rac1, however,decreased the number of AChR full-sized clusters but increasedthose of microclusters (Fig. S3B–E). This result is consistent with ourprevious observations that prolonged activation of Rac1 after the

Fig. 2. Patterns and localization of activatedRac1 during AChR clustering. (A) C2C12myotubes were treated with agrin for 1, 2, 4, 8 or16 h, AChR clusters were stained by AlexaFluor555-conjugated α-BTX. Scale bar: 50 μm.(B) Quantification analysis of AChR cluster(≥5 μm2) and microcluster (<5 μm2) numbers.(C) Agrin induces transient activation of Rac1.C2C12 myotubes were treated with agrin for0–60 min, active Rac1 was pulled down by GSTfused to PBD. (D) Myotubes were transfected withYFP-PBD mRNA together with Rac1-WT or T17NmRNA. Myotubes were fixed after they had beentreated for 1.5 h with agrin. AChR clusters werestained with Alexa Fluor555-conjugated α-BTX.Arrowheads indicate clusters of activated Rac1(green fluorescence of YFP-PBD that did notcolocalize with AChR clusters), arrow indicatesclusters of activated Rac1 colocalized with AChRclusters. Scale bar: 20 μm.

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initial phase of AChR clustering impairs the formation of full-sizedclusters. We then studied how alterations in Rac1 activity affectAChR stability. Myotubes were treated with agrin for 8 h, and AChRclusters were labeled with Alexa Fluor555-conjugated α-BTX. Rac1-WT or Rac1-T17N was then introduced to myotubes to study thedispersal of these ‘pre-existing’ AChR clusters (Fig. 4A). Notably,Rac1 activation caused accelerated removal of AChR clusters,indicated by the reduced number of full-sized clusters (Fig. 4B–D).We also quantified larger size (>20 μm2) full-sized clusters and foundthat Rac1 activation decreased the area of these clusters (Fig. 4E).Rac1-T17N, however, caused more clusters of all sizes to be retainedafter agrin removal (Fig. 4B–E). Moreover, we labeled pre-existingclusters with a saturating dose of Alexa Fluor555-conjugated α-BTX.Unbound α-BTX was then washed off and the myotubes were re-incubated with agrin for another 12 h, followed by a secondincubation with Alexa Fluor488-conjugated α-BTX (Fig. 4F). Asα-BTX binds to AChRs in a relatively irreversible fashion, this two-round labeling method can be used to distinguish between pre-existing and newly formed clusters (Fig. S4) (Kummer et al., 2004).On the one hand, we observed that inhibition of Rac1 had no effecton pre-existing clusters but significantly reduced newly formedclusters (Fig. 4G–J). On the other hand, Rac1 activation decreased thenumber of pre-existing clusters but increased that of newly formedmicroclusters (Fig. 4G–J). Consistently, after Rac1 activation, the

total cluster area was composed of fewer pre-existing clusters andmore newly formed clusters (Fig. 4K). Together, these results suggestthat Rac1 inhibition is important for the stabilization of pre-existingclusters, whereas Rac1 activation contributes to the dispersal andturnover of AChR clusters.

Pak1 mediates myotube fusion and destabilization of AChRclusters induced by Rac1 activationPak1, one of the most-studied downstream effectors of Rac1, has beensuggested to be involved in the regulation of myotube formation andAChR clustering (Joseph et al., 2017; Luo et al., 2002). We found thatPak1 activity, indicated by its phosphorylation at Ser199 and Ser204,is tightly correlated with Rac1 activity (Fig. 5A). We then askedwhether Rac1-induced myotube fusion and AChR cluster dispersalwere mediated by Pak1. Indeed, the dominant-negative K298Rmutation of Pak1 (Pak1-DN), which blocks Pak1 activity, markedlyreversed the myotube fusion defects caused by Rac1-G12V(Fig. 5B,C). Pharmacological inhibition of actin polymerizationwith latrunculin A also inhibited Rac1-G12V-inducedmyotube fusion(Fig. 5D,E), suggesting that myotube fusion caused byRac1 activationis mediated by Pak1 activation and actin polymerization. However,expression of Pak1-WT or of the constitutively active T422E Pak1mutant (Pak1-CA) did not induce myotube fusion (Fig. S5),suggesting the involvement of other Rac1 effectors in this process.

Fig. 3. Initial formation of AChR clustersrequires Rac1 activation. (A) Schematicshowing the examination of AChR clusteringinduced by treatment with agrin for 1.5 or 8 h,followed by labeling with Alexa Fluor-conjugated α-BTX. (B) Representative imagesshowing AChR clusters at 1.5 h. Insets (topright corner) aremagnifications of boxed areasshowing small clusters and microclusters.(C) Number of AChR microclusters (<5 μm2).*P<0.05, Rac1-WT (WT) or Rac1-T17N(T17N) vs control (Ctrl); ###P<0.001, T17N vsWT. (D) Numbers of AChR clusters (≥5 μm2).*P<0.05, T17N vs Ctrl; #P<0.05, T17N vs WT.(E) Area of AChR clusters. (F) Representativeimages showing AChR clusters after 8 h oftreatment with agrin. (G–I) Quantification ofnumbers of AChR microclusters (G), numberof clusters (H) and cluster area (I). *P<0.05,**P<0.01,WTor T17N vsCtrl; ##P<0.01, T17NvsWT. All data are presented as mean±s.e.m.from at least three independent experiments.Statistical analysis was subjected to one-wayANOVA with Bonferroni multiple comparisontest. All scale bars: 50 μm.

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Blocking the activation of the Arp2/3 complex – a downstreameffector of Rac1 – by Wiskostatin failed to inhibit myotube fusion(Fig. 5D,E). To study the involvement of Pak1 in Rac1-mediateddispersal of pre-existing AChR clusters, we co-transfected Rac1-WTand Pak1-DN into myotubes after agrin had been added for 8 h andremoved. Pak1 inhibition, indeed, blocked Rac1-mediated AChRdestabilization (Fig. 5F,G). Therefore, Rac1-induced myotube fusionand AChR destabilization are largely mediated by Pak1.

Several GEFs and GAPs that activate Rac1 participate in theregulation of AChR clusteringWe have shown that, in mature myotubes, Rac1 is generallyinhibited, but that it is transiently activated during the formation ofAChR clusters. Moreover, Rac1 is activated occasionally on themyotube surface and surrounds AChR clusters. Therefore, a precisespatiotemporal regulation of Rac1 activity is required to control thenormal development of myotubes and AChR clusters. However, sofar it is unknown which Rac1 GEFs or GAPs participate in theregulation of AChR clustering. To this end, we selected several Rac1

GEFs and GAPs that are expressed in the skeletal muscle, includingTiam1, Tiam2, Kalirin, Trio, α-PIX (ARHGEF6), β-PIX(ARHGEF7), Dock1, Dock4, Dock7, Vav1, Vav2, α2-chimaerin(CHN1), β2-chimaerin (CHN2) and BCR. To target individualfactors, siRNAs were designed, whose knockdown efficiency wasconfirmed by real-time quantitative RT-PCR to be >60% (Fig. S6).We transfected each siRNA into myotubes and added agrin toinduce AChR clustering. Interestingly, knockdown of α2-chimaerin, BCR, Kalirin, Trio, β-PIX, Dock1 and Vav1 led todecreased AChR clustering, whereas knockdown of Vav2 promotedAChR clustering (Fig. 6A–C). These results indicate that Rac1 iscooperatively regulated by multiple GAPs and GEFs during agrin-induced clustering of AChR.

DISCUSSIONOur study showed that, although Rac1 activation is required foragrin-induced AChR cluster formation, persistent activation of Rac1leads to destabilization of AChR clusters; and constitutive activationof Rac1 even causes myotube fusion. Both myotube fusion and

Fig. 4. Stabilization of AChR clusters depends onRac1 inhibition. (A) Schematic showing themanipulationof Rac1 activity at the AChR cluster dispersal phase(treatment with agrin for 8 h, followed by no treatment for12 h), followed by labeling with Alexa Fluor-conjugatedα-BTX. (B) Rac1 activation accelerates AChR clusterdispersal. (C–E) Quantification of AChR microclusternumber (C), cluster number (D) and cluster area (E).*P<0.05, **P<0.01, Rac1-WT (WT) or Rac1-T17N (T17N)vs control (Ctrl); #P<0.05, ###P<0.001, Rac1-T17N vsWT.(F) Schematic showing the examination of Rac1 activityon both pre-existing and newly formed clusters (treatmentwith agrin for 8 h, followed by no treatment for 12 h),followed by labeling with Alexa Fluor555-conjugatedα-BTX. (G) Representative images showing pre-existingclusters labeled with Alexa Fluor555-conjugated α-BTX,and newly formed clusters labeled with Alexa Fluor488-conjugated α-BTX. (H,I) For each population of pre-existing or newly formed clusters, AChR microclusternumber (H), cluster number (I) and cluster area (J) werequantified. (K) Quantification of pre-existing, newly formedand intermingled (containing both pre-existing and newlyformed) clusters in percent. *P<0.05, **P<0.01,***P<0.001, Rac1-WT or T17N vs Ctrl; #P<0.05,##P<0.01, ###P<0.001, Rac1-T17N vs WT. All data arepresented as mean±s.e.m. from at least threeindependent experiments. Statistical analysis wassubjected to one-way ANOVA with Bonferroni multiplecomparison test. All scale bars: 50 μm.

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AChR dispersal induced by Rac1 are dependent on Pak1 activation.The fact that inhibition of Rac1 hardly alters myotube morphologyand AChR dispersal suggests that Rac1 activity is largely inhibitedand not required for the terminal differentiation of myotubes andthe maintenance of AChR clusters. Two Rac1 GAPs and Six Rac1GEFs were found to be important for normal AChR clustering.Together, our study reveals that Rac1 is tightly regulated to ensureand control normal AChR clustering.It has been shown previously that Rac1 activation at the onset of

myoblast differentiation is required for myoblast fusion (Charrasseet al., 2007). Rac1 is involved in the organization of the lipid raft-containing fusion sites in myogenic cells (Mukai and Hashimoto,2013). During the process of myotube formation, however, Rac1activity is gradually decreased (Charrasse et al., 2007). Consistentwith these findings, we have shown that inhibition of Rac1 in well-developed myotubes does not affect myotube morphology but thatconstitutive activation of Rac1 induces excessive myotube fusion.This suggests that, although activation of Rac1 mediates the initialmyoblast fusion, it generally needs to be inhibited to shut down thefusion process once myotubes are properly formed. Nonetheless,Rac1 is not completely quiescent; and its active form is sparselyobserved at the surface of mature myotubes. Therefore, maturemuscle fibers may be still fusion-competent in order to remodel

during muscle growth or regeneration. Here we demonstrated thatPak1 and actin polymerization are important to mediate the myotubefusion induced by Rac1. However, Pak1 alone is insufficient tocause myotube fusion. It would, therefore, be interesting to explorethe involvement of other Rac1 effectors.

For the formation and maintenance of AChR clusters, the tightregulation of Rac1 activity is required. Rac1 activation, transientlyinduced by agrin, facilitates the initial phase of AChR clusterformation. We observed that Rac1-WT induces more microclustersshortly after treatment with agrin, whereas Rac1-T17N inhibits theformation of both microclusters and full-sized clusters (Fig. 7A,B).This is consistent with previous evidence that Rac1 activationleads to AChR microcluster formation (Weston et al., 2003, 2000).However, when examined in response to prolonged treatment withagrin (8 h), Rac1 activation decreases the number of full-sizeclusters and increases the number of microclusters, whereas Rac1inhibition decreases the number of full-size clusters but does nottrigger microcluster formation (Fig. 7A,B). This suggests that Rac1activation promotes the initial formation of AChR clusters, but theseclusters can be easily dispersed unless Rac1 is subsequentlyinhibited. Indeed, Rac1 activation accelerates the dispersal of pre-existing AChR clusters no matter whether agrin is present or not,and Rac1 activation also promotes replacement of pre-existing

Fig. 5. Pak1 mediates Rac1 activity on myotube fusionand AChR cluster destabilization. (A) Levels of Pak1phosphorylated at Ser199 and Ser204 (pS199/204-Pak1)were tightly correlated with the amount of active Rac1.Rac1-WT, Rac1-T17N or different amounts of Rac1-G12Vwere transfected into C2C12 myotubes; phosphorylationof Pak1 was examined by western blotting. (B) Inhibition ofPak1 reverses myotube fusion upon expression of Rac1-G12V. Rac1-G12V alone or together with Pak1-DN wastransfected into myotubes, anti-MHC antibody was usedto stain myotubes. (C) Quantification of myotube width.***P<0.001, Rac-G12V vs Ctrl; #P<0.05, Rac1-G12V+Pak1-DN vs Rac1-G12V. (D) Myotube fusion induced byRac1 hyperactivation was blocked upon addition of theactin polymerization inhibitor latrunculin A (Lat) or but notthe Arp2/3 inhibitor. Myotubes were transfected withRac1-G12V and treated with the actin polymerizationinhibitor latrunculin A (Lat) or the Arp2/3 inhibitorWiskostatin (Wis). (E) Quantification of myotube width.***P<0.001, Rac1-G12V only or with Wis vs Ctrl;###P<0.001, G12V+Lat vs G12V; &&&P<0.001, Rac1-G12V+Wis vs G12V. (F) Pak1 inhibition restores stabilityof AChR clusters in Rac1-G12V-expressing myotubes.Myotubes were treated with agrin for 8 h, AChR clusterswere labeled, and Rac1-G12V alone or together withPak1-DN were transfected into myotubes. The myotubeswere continuously cultured without agrin for 12 h.(G) Number of pre-existing AChR clusters was quantified.*P<0.05, Rac1-WT vs Ctrl; #P<0.05, Rac1-WT+Pak1-DNvs Rac1-WT. All data shown in this figure are presented asmean±s.e.m. from at least three independentexperiments. Statistical analysis was subjected to one-way ANOVA with Bonferroni multiple comparison test. Allscale bars: 50 μm.

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clusters with newly formed ones. It has been shown previously thatthe endplate AChR clusters (i.e. AChR clusters at the postsynapticmuscle membrane of the neuromuscular junction) are relativelystable and have a long half-life, but recycling and intra-junctionalmigration of receptors are important processes that contribute tocluster dynamics (Akaaboune et al., 1999, 2002; Bruneau et al.,2005). In support with this notion, we found that active Rac1 rarelycolocalizes with high-density AChR clusters; instead it is mainlypresent in close proximity to the clusters. Therefore, we propose that

Rac1 activation, by promoting removal of pre-existing clustersand formation of new microclusters, regulates the dynamicsand recycling of AChR clusters. The formation, refinement,maintenance and dispersal of AChR clusters may be tightlycontrolled by local activation or inhibition of Rac1.

Since Rac1 has to be accurately regulated to determine myotubedevelopment and AChR clustering, a complex mechanism forspatiotemporal Rac1 activity control must be involved. For thisstudy, we screened 14 Rac1 GEFs and GAPs, and identified 8

Fig. 6. Several Rac1 GEFs and Rac1 GAPs participatein the regulation of AChR clustering. (A) siRNAs ofdifferent Rac1GEFs andRac1GAPswere transfected intoC2C12myotubes. Agrin was added for 8 h to induce AChRclustering. Scale bar: 50 μm. (B,C) Quantification of AChRcluster number (B) and area (C) after siRNA transfection ofGEFs and GAPs normalized to those transfected withcontrol siRNA (Ctrl). *P<0.05, **P<0.01, ***P<0.001,Student’s t-test. Data are presented as mean±s.e.m. fromat least three independent experiments.

Fig. 7. A summary of the findings in this study.(A) Influences of Rac1 activation and inhibition onAChR cluster formation and stabilization atdifferent stages. (B) A proposed pattern ofchanges of Rac1 activity, AChRmicroclusters andfull-sized clusters during the course of agrin-induced AChR clustering. (C) Rac1 can beregulated by several GEFs and GAPs duringAChR clustering.

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that participate in AChR clustering, including the Rac1 GAPsα2-chimaerin and BCR, and the Rac1 GEFs Kalirin, Trio, β-PIX,Dock1, Vav1 and Vav2. To our knowledge, this is the first time thatthis set of Rac1 regulators has been reported to have functional rolesin AChRclustering in skeletal muscle (Fig. 7C). Interestingly, Vav2 isthe only Rac1 regulator that appears to have opposite roles comparedwith other identified regulators. Knockdown of Vav2 leads to thepromotion of AChR clusters, whereas knockdown of either of theother 7 regulators always decreased the number of AChR clusters.Additional work regarding the expression and activation patterns ofthese Rac1 regulators is required to understand the action of eachregulator and their cooperative modulation of Rac1 activity.

MATERIALS AND METHODSReagents, antibodies and constructsAnti-Rac1 antibody was purchased from BD Biosciences (Cat# 610651;1:2000 for western blotting), anti-MHC (anti-myosin heavy chain) antibodywas from Millipore (Cat# 05-716; 1:4000 for immunocytochemistry), anti-α-tubulin antibody was from Sigma-Aldrich (Cat# T6074; 1:5000 forwestern blotting), antibodies against Pak1 (Cat# 2602; 1:2000 for westernblotting) and phosphorylated Pak1 (at Ser199 and Ser204; Cat# 2605;1:1000 for western blotting) were from Cell Signaling Technology. AlexaFluor555 and Alexa Fluor488 conjugated to the neurotoxin α-BTX thatlabels AChRs were from Life Technologies. Recombinant rat C-terminalagrin was from R&D Systems. Latrunculin A and Wiskostatin were fromMillipore. YFP-PBD plasmid was a gift from Joel Swanson (Addgeneplasmid #11407) (Hoppe and Swanson, 2004). Mouse Rac1-WT, Rac1-T17N (dominant negative) and Rac1-G12V (constitutively active) plasmidshave been described previously (IP et al., 2012; Xiao et al., 2013), and Rac1-Q61L (constitutively active) was generated by mutagenesis. Mouse Pak1was cloned from mouse brain cDNA by RT-PCR and subcloned intopcDNA3.0 vector. Dominant negative Pak1-DN (K298R) and constitutivelyactive Pak1-CA (T422E) mutants were generated by mutagenesis. Theprimers used for plasmid construction are shown in Table S1.

Cell culture and transfectionMouse C2C12 myoblasts (ATCC) were cultured under 5% CO2 and 95% airat 37°C as previously described (Shi et al., 2010a). Briefly, C2C12 myoblastswere maintained in Dulbecco’s modified Eagle’s medium (DMEM; LifeTechnologies) containing 20% fetal bovine serum (Life Technologies).Myotube differentiation was induced when myoblasts had grown to 100%confluency, growth medium was then switched to differentiation medium(DMEM supplemented with 2% horse serum, Life Technologies). Inductionof AChR clustering was performed at day 3.5 of differentiation. Forexpression of YFP-PBD, Rac1, Pak1 or their mutant forms, we generatedmRNAs by in vitro transcription using the mMESSAGE mMACHINE T7Ultra Kit (Life Technologies). Respective mRNA (normally 0.5 μg, but 0.02–0.5 μg for Rac1-G12V and Rac1-Q61L) was transfected into C2C12myotubes using Lipofectamine 2000 (Life Technologies). To study AChRformation and dispersal, mRNA was transfected 3 h before treatment withagrin, 1 h after agrin treatment or immediately after agrin had been removed,according to the experimental designs. For siRNA knockdown experiments,C2C12 myotubes were transfected with 40 nmol siRNA using Lipofectamine2000 (Invitrogen) on day 2.5 of differentiation.

AChR clustering assay and myotube morphology analysisFor agrin-induced AChR clustering, C2C12 myotubes were treated with10 ng/ml agrin for 1–16 h. AChR clusters were labeled in live myotubesusing Alexa Fluor555-conjugated α-BTX. To examine the stability of AChRclusters, myotubes were treated with agrin for 8 h, and the AChR clusterswere labeled by Alexa Fluor555-conjugated α-BTX as pre-existing clusters.Agrin was then washed off and myotubes were cultured in agrin-freemedium for another 12 h. Alternatively, agrin was added again for another12 h, and Alexa Fluor488-conjugated α-BTX was used to label newlyformed AChR clusters. Myotubes were fixed using 4% paraformaldehyde at

room temperature for 20–30 min. To analyze myotube fusion, anti-MHCantibody was used to stain the myotubes. F-actin was labeled byRhodamine-phalloidin. Images of AChR clusters were captured by usinga 40× objective and an ImagerA2 fluorescence microscope (Carl Zeiss AG).The myotubewidth, number and area of AChR clusters were quantified from10 random fields using ImageJ software. Cluster areas of >5 μm2 werecounted as full-sized clusters, whereas those of <5 μm2 were counted asmicroclusters. To analyze the fluorescence intensity of Alexa Fluor 555- and488-conjugated α-BTX-labeled AChR clusters, Imaris 9.1.2 software wasused. Briefly, a 2D rebuild surface that exactly draws out the clusters wascreated for each image by adjusting the values of Smooth and Thresholdunder the Surpass module. The average intensity, area and number ofclusters were quantified.

Rac1 activity assayRac1 activity assay was carried out as described previously (Shi et al., 2007).Briefly, myotube proteins were harvested using a lysis buffer containing50 mM Tris pH 7.2, 1% Triton X-100, 500 mM NaCl, 0.1% SDS, 0.5%sodium deoxycholate, 10 mM MgCl2, 1 mM PMSF, 10 g/ml leupeptin and10 g/ml aprotinin. GTP-Rac1 was pulled down by binding to glutathione-coupled agarose beads that had been conjugated to PBD-fused GST (GST-PBD) for 90 min at 4°C. The agarose beads were washed 3× with Tris bufferpH 7.2 (50 mM), containing 1% Triton X-100, 10 mM MgCl2, 150 mMNaCl, 0.1 mM PMSF, 10 g/ml leupeptin and 10 g/ml aprotinin and thenresuspendedwith 2× Laemmli sample buffer (Santa Cruz Biotechnology) andsubjected to western blot analysis.

Knockdown of Rac1 GEFs and Rac GAPs in C2C12 myotubesFour siRNAs were designed and synthesized for each gene by QIAGEN, theknockdown efficiencies were tested using quantitative RT-PCR. Thesequences of the most effective siRNAs for individual genes are shown inTable S1.

Quantitative RT-PCRTotal RNA from C2C12 myotubes was isolated by using the RNAiso Pluskit (TaKaRa) following the manufacturer’s suggested procedure. RT-PCRwas performed as previously reported (Liao et al., 2018; Xiang et al., 2016).In brief, reverse transcription was performed to obtain cDNA products usingM-MLV reverse transcriptase (Promega) and oligo(dT) primers. cDNAswere then mixed with iQ SYBR® Green (Bio-Rad) and qPCR wasperformed using a LightCycler 480 (Roche). Each PCR reaction wasperformed in triplicates and the GAPDH gene was used as an internalstandard. The primers used for RT-PCR are shown in Table S1.

Statistical analysisData were presented as mean±standard error of mean (±s.e.m.) from at leastthree independent experiments, and analyzed by using GraphPad Prism 5software. Statistically significant differences between two groups wereanalyzed by Student’s t-test. Significant differences between multiplegroups were determined by one-way ANOVA followed by BonferroniMultiple Comparison Test.

AcknowledgementsWe thank Dr Yuewen Chen and Miss Yumei Liao for their excellent technicalassistance. We also thank Dr Yibo Qu, Dr Lisheng Peng, and Miss Xiaoxiao Li forsharing reagents. This work was supported in part by the National Natural ScienceFoundation of China [grant number 31471046]. L.S. is funded by The Academy ofMedical Sciences Newton Advanced Fellowship in partnership with The RoyalSociety and The National Natural Science Foundation of China [UK/China grantnumbers: NA160314/81761130084].

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: L.S.; Methodology: Y.B., Y.P., J.X., L.S.; Validation: Y.B., D.G.,X.S., G.T., T.L.; Formal analysis: Y.B.; Investigation: Y.B., D.G., X.S., G.T., T.L., L.S.;

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Data curation: Y.B.; Writing - original draft: Y.B., L.S.; Writing - review & editing: Y.B.,L.S.; Supervision: L.S.; Project administration: L.S.; Funding acquisition: L.S.

FundingThis work was supported in part by the National Natural Science Foundation ofChina [grant number: 31471046]. L.S. is funded by The Academy of MedicalSciences Newton Advanced Fellowship in partnership with the Royal Society andthe National Natural Science Foundation of China [UK/China grant numbers:NA160314 and 81761130084].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.215251.supplemental

ReferencesAkaaboune, M., Culican, S. M., Turney, S. G. and Lichtman, J. W. (1999). Rapidand reversible effects of activity on acetylcholine receptor density at theneuromuscular junction in vivo. Science 286, 503-507.

Akaaboune, M., Grady, R. M., Turney, S., Sanes, J. R. and Lichtman, J. W.(2002). Neurotransmitter receptor dynamics studied in vivo by reversible photo-unbinding of fluorescent ligands. Neuron 34, 865-876.

Bai, Y., Xiang, X., Liang, C. and Shi, L. (2015). Regulating Rac in the nervoussystem: molecular function and disease implication of Rac GEFs and GAPs.Biomed. Res. Int. 2015, 632450.

Basu, S., Sladecek, S., Martinez de la Pen a y Valenzuela, I., Akaaboune, M.,Smal, I., Martin, K., Galjart, N. and Brenner, H. R. (2015). CLASP2-dependentmicrotubule capture at the neuromuscular junction membrane requires LL5betaand actin for focal delivery of acetylcholine receptor vesicles. Mol. Biol. Cell 26,938-951.

Bruneau, E., Sutter, D., Hume, R. I. and Akaaboune, M. (2005). Identification ofnicotinic acetylcholine receptor recycling and its role in maintaining receptordensity at the neuromuscular junction in vivo. J. Neurosci. 25, 9949-9959.

Charrasse, S., Comunale, F., Fortier, M., Portales-Casamar, E., Debant, A. andGauthier-Rouviere, C. (2007). M-cadherin activates Rac1 GTPase through theRho-GEF trio during myoblast fusion. Mol. Biol. Cell 18, 1734-1743.

Dai, Z., Luo, X., Xie, H. and Peng, H. B. (2000). The actin-driven movement andformation of acetylcholine receptor clusters. J. Cell Biol. 150, 1321-1334.

Dobbins, G. C., Zhang, B., Xiong, W. C. and Mei, L. (2006). The role of thecytoskeleton in neuromuscular junction formation. J. Mol. Neurosci. 30, 115-118.

Gautam, M., Noakes, P. G., Mudd, J., Nichol, M., Chu, G. C., Sanes, J. R. andMerlie, J. P. (1995). Failure of postsynaptic specialization to develop atneuromuscular junctions of rapsyn-deficient mice. Nature 377, 232–236.

Gawor, M. and Proszynski, T. J. (2018). The molecular cross talk of the dystrophin-glycoprotein complex. Ann. N. Y. Acad. Sci. 1412, 62-72.

Henriquez, J. P., Webb, A., Bence, M., Bildsoe, H., Sahores, M., Hughes, S. M.and Salinas, P. C. (2008). Wnt signaling promotes AChR aggregation at theneuromuscular synapse in collaboration with agrin. Proc. Natl. Acad. Sci. USA105, 18812-18817.

Hoppe, A. D. and Swanson, J. A. (2004). Cdc42, Rac1, and Rac2 display distinctpatterns of activation during phagocytosis. Mol. Biol. Cell 15, 3509-3519.

Ip, J. P. K., Shi, L., Chen, Y., Itoh, Y., Fu, W.-Y., Betz, A., Yung, W.-H., Gotoh, Y.,Fu, A. K. Y. and Ip, N. Y. (2012). alpha2-chimaerin controls neuronal migrationand functioning of the cerebral cortex through CRMP-2. Nat. Neurosci. 15, 39-47.

Jacobson, C., Cote, P. D., Rossi, S. G., Rotundo, R. L. and Carbonetto, S.(2001). The dystroglycan complex is necessary for stabilization of acetylcholinereceptor clusters at neuromuscular junctions and formation of the synapticbasement membrane. J. Cell Biol. 152, 435-450.

Joseph, G. A., Lu, M., Radu, M., Lee, J. K., Burden, S. J., Chernoff, J. andKrauss, R. S. (2017). Group I Paks promote skeletal myoblast differentiation invivo and in vitro. Mol. Cell. Biol. 37, e00222-16.

Kumari, S., Borroni, V., Chaudhry, A., Chanda, B., Massol, R., Mayor, S. andBarrantes, F. J. (2008). Nicotinic acetylcholine receptor is internalized via a Rac-

dependent, dynamin-independent endocytic pathway. J. Cell Biol. 181,1179-1193.

Kummer, T. T., Misgeld, T., Lichtman, J. W. and Sanes, J. R. (2004). Nerve-independent formation of a topologically complex postsynaptic apparatus. J. CellBiol. 164, 1077-1087.

Lee, C. W., Han, J., Bamburg, J. R., Han, L., Lynn, R. and Zheng, J. Q. (2009).Regulation of acetylcholine receptor clustering by ADF/cofilin-directed vesiculartrafficking. Nat. Neurosci. 12, 848-856.

Liao, Y., Zhuang, X., Huang, X., Peng, Y., Ma, X., Huang, Z.-X., Liu, F., Xu, J.,Wang, Y., Chen, W.-M. et al. (2018). A bivalent securinine compound SN3-L6induces neuronal differentiation via translational upregulation of neurogenictranscription factors. Front. Pharmacol. 9, 290.

Linnoila, J., Wang, Y., Yao, Y. and Wang, Z.-Z. (2008). A mammalian homolog ofDrosophila tumorous imaginal discs, Tid1, mediates agrin signaling at theneuromuscular junction. Neuron 60, 625-641.

Luo, Z. G., Wang, Q., Zhou, J. Z., Wang, J., Luo, Z., Liu, M., He, X., Wynshaw-Boris, A., Xiong, W. C., Lu, B. et al. (2002). Regulation of AChR clustering byDishevelled interacting with MuSK and PAK1. Neuron 35, 489-505.

Mukai, A. and Hashimoto, N. (2013). Regulation of pre-fusion events: recruitmentof M-cadherin to microrafts organized at fusion-competent sites of myogenic cells.BMC Cell Biol. 14, 37.

Nizhynska, V., Neumueller, R. and Herbst, R. (2007). Phosphoinositide 3-kinaseacts through RAC and Cdc42 during agrin-induced acetylcholine receptorclustering. Dev. Neurobiol. 67, 1047-1058.

Proszynski, T. J., Gingras, J., Valdez, G., Krzewski, K. and Sanes, J. R. (2009).Podosomes are present in a postsynaptic apparatus and participate in itsmaturation. Proc. Natl. Acad. Sci. USA 106, 18373-18378.

Rossman, K. L., Der, C. J. and Sondek, J. (2005). GEFmeans go: turning on RHOGTPases with guanine nucleotide-exchange factors. Nat. Rev. Mol. Cell Biol. 6,167-180.

Sanes, J. R. and Lichtman, J. W. (1999). Development of the vertebrateneuromuscular junction. Annu. Rev. Neurosci. 22, 389-442.

Sanes, J. R. and Lichtman, J. W. (2001). Induction, assembly, maturation andmaintenance of a postsynaptic apparatus. Nat. Rev. Neurosci. 2, 791-805.

Shi, L., Fu, W.-Y., Hung, K.-W., Porchetta, C., Hall, C., Fu, A. K. and Ip, N. Y.(2007). Alpha2-chimaerin interacts with EphA4 and regulates EphA4-dependentgrowth cone collapse. Proc. Natl. Acad. Sci. USA 104, 16347-16352.

Shi, L., Butt, B., Ip, F. C. F., Dai, Y., Jiang, L., Yung, W.-H., Greenberg, M. E., Fu,A. K. Y. and Ip, N. Y. (2010a). Ephexin1 is required for structural maturation andneurotransmission at the neuromuscular junction. Neuron 65, 204-216.

Shi, L., Fu, A. K. Y. and Ip, N. Y. (2010b). Multiple roles of the Rho GEF ephexin1 insynapse remodeling. Commun. Integr. Biol. 3, 622-624.

Shi, L., Fu, A. K. Y. and Ip, N. Y. (2012). Molecular mechanisms underlyingmaturation and maintenance of the vertebrate neuromuscular junction. TrendsNeurosci. 35, 441-453.

Tcherkezian, J. and Lamarche-Vane, N. (2007). Current knowledge of the largeRhoGAP family of proteins. Biol. Cell 99, 67-86.

Weston, C., Yee, B., Hod, E. and Prives, J. (2000). Agrin-induced acetylcholinereceptor clustering is mediated by the small guanosine triphosphatases Rac andCdc42. J. Cell Biol. 150, 205-212.

Weston, C., Gordon, C., Teressa, G., Hod, E., Ren, X.-D. and Prives, J. (2003).Cooperative regulation by Rac and Rho of agrin-induced acetylcholine receptorclustering in muscle cells. J. Biol. Chem. 278, 6450-6455.

Wu, H., Xiong, W. C. and Mei, L. (2010). To build a synapse: signaling pathways inneuromuscular junction assembly. Development 137, 1017-1033.

Xiang, X., Li, S., Zhuang, X. and Shi, L. (2016). Arhgef1 negatively regulatesneurite outgrowth through activation of RhoA signaling pathways. FEBS Lett. 590,2940-2955.

Xiao, Y., Peng, Y., Wan, J., Tang, G., Chen, Y., Tang, J., Ye, W.-C., Ip, N. Y. andShi, L. (2013). The atypical guanine nucleotide exchange factor Dock4 regulatesneurite differentiation through modulation of Rac1 GTPase and actin dynamics.J. Biol. Chem. 288, 20034-20045.

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